On 25/09/2010 23:25, DK wrote:
>
> Thanks for the details. All seem reasonable. One nitpick, perhaps
> will be helpful in the future: don't bother with anti-peptide Ab unless
> functional inhibition studies are planned. It's alwasy easier to make
> small fragment in E.coli, immunize with denatured protein and get
> super pure polyclonals for Western by purifying against blotted
> protein.
A consideration I hadn't explained is that I'm distinguishing between an
X gene and an autosomal relative, so a peptide antibody was necessary to
direct it to regions of greatest divergence between the two. As for the
ease or otherwise of expressing proteins in E. coli, that's kind of the
problem here!
>
> OK, things I can think of off hand:
> - The gene produces super-unstable mRNA.
> - The gene, somewhere near the start, has termination signal
> (either transcription or translation; the former occasionally
> happens for termination).
>
> So my first impulse would be to see if the message is even made.
As I said in the previous post, for the most recent construct (in
pT7CFE1 vector) we did indeed isolate/sequence the mRNA and confirm it's
expressed correctly in one of our in vitro systems (T7 transcription
coupled with rabbit reticulocyte lysate). For this vector, the RBS,
transcription initiation site and first half dozen amino acids are
supplied by the vector, so we can also rule out a problem with
transcriptional initiation.
>
> If with a totally deifferent gene sequence you continue to observe all
> the same, get all kinds of more preliminary data and start get
> writing a grant. :-)
Current working hypothesis is that there's something near the start of
the ORF that causes the problem. Could be a non-standard translation
termination signal, could be mRNA structure, could be a run of
non-preferred codons for which the tRNA is more abundant in spermatids,
could be some problem in the protein domain structure.... or any number
of other options I've not thought of yet!
Interestingly, the IHC signal alters its localisation as germ cells
mature - it's cytoplasmic in round spermatids and much more localised in
elongating spermatids. In whole testis Western the 55 kDa band comes up
in both the RIPA-soluble and insoluble fractions. I suspect that the
cytoplasmic signal is the soluble form and the localised signal is the
insoluble form. Westerns next week should help resolve this :-)
If I'm right about the soluble/insoluble forms, we can hypothesise that
there is a specific chaperone protein in round spermatids that keeps the
monomers in a soluble form until they're ready for use. In the absence
of that factor, maybe it starts clumping up and dropping out of solution
in our in vitro / culture systems. The E.coli expression experiments
did give a load of insoluble crud - absolutely *huge* pellet after lysis
with BugBuster - whatever it was didn't however have a His-tag on it
(C-terminal) and didn't react with the peptide antibody (also C-terminal).
I think what I'm going to do is try cloning just the second half of the
protein - only requires ordering 1 more forward primer and a few days'
PCR/cloning work. I'd really like to do a yeast 2-hybrid screen further
down the line though, so expressing as much of the protein as possible
would be very helpful. On the other hand, if the interaction domain(s?)
mean it can't be expressed in yeast, we're a bit stuffed on that front...
There's also the Hail Mary pass option of spiking testis extract into
the in vitro translation reaction and hoping it contributes whatever
factor is necessary. I guess I could homogenise testis tissue in a
buffer of choice (RIPA buffer or PBS? Which one won't inhibit the in
vitro reactions?) and just stick some in. Or I could do a collagenase
digestion of the tissue, spin down the separated cells and actually make
a testis lysate to use in place of the reticulocyte lysate. The latter
especially seems like something that would take a lot of time and money
to optimise though :-(
Any further suggestions?
Peter